Microneedles for Transdermal Biosensing: Current Picture and Future Direction

A novel trend is rapidly emerging in the use of microneedles, which are a miniaturized replica of hypodermic needles with length-scales of hundreds of micrometers, aimed at the transdermal biosensing of analytes of clinical interest, e.g., glucose, biomarkers, and others. Transdermal biosensing via microneedles offers remarkable opportunities for moving biosensing technol-ogies and biochips from research laboratories to real-fi eld applications, and envisages easy-to-use point-of-care microdevices with pain-free, minimally invasive, and minimal-training features that are very attractive for both devel-oped and emerging countries. In addition to this, microneedles for trans-dermal biosensing offer a unique possibility for the development of biochips provided with end-effectors for their interaction with the biological system under investigation. Direct and effi cient collection of the biological sample to be analyzed will then become feasible in situ at the same length-scale of the other biochip components by minimally trained personnel and in a minimally invasive fashion. This would eliminate the need for blood extraction using hypodermic needles and reduce, in turn, related problems, such as patient infections, sample contaminations, analysis artifacts, etc. The aim here is to provide a thorough and critical analysis of state-of-the-art developments in this novel research trend, and to bridge the gap between microneedles and biosensors

Lab-on-a-chip platforms for pathogen analysis

Infectious diseases caused by pathogenic microorganisms are a big burden in developed and developing countries. The emergence and rapid global spread of virus and antimicrobial resistant bacteria is a significant threat to patients, healthcare systems and the economy of countries. Early pathogen detection is often hampered by low concentrations present in complex matrices such as food and body fluids.Microfluidic technologies offer new and improved approaches for detection of pathogens on the microscale. Here, two microfluidic platforms for pathogen sorting and molecular identification were investigated: (1) inertial focusing and (2) microscale immiscible filtration. Inertial focusing in two serpentine channel designs etched in glass at different depths was evaluated with different microparticles, bacteria and blood. The shallow design allowed 2.2-fold concentration of Escherichia coli O157 cells, whereas the deep design accomplished recovery of 54% E. coli O157 depleted from 97% red blood cells in 0.81% haematocrit at flowrates of 0.7 mL min-1.A lab-on-a-chip platform based on microscale immiscible filtration was investigated for capture and detection of nucleic acids and bacteria. For nucleic acids, oligo (dT) functionalised magnetic beads or silica paramagnetic particles in GuHCl were used to capture genomic RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and genomic DNA from Neisseria gonorrhoeae, respectively. On-chip amplification and detection were performed via colorimetric loop-mediated isothermal amplification (LAMP). Results showed sensitive and specific detection of targeted nucleic acids (470 RNA copies mL-1 and 5 × 104 DNA copies mL-1) with no cross-reactivity to other RNAs and DNAs tested. The whole workflow was integrated in a single device and time from sample-in to answer-out was within 1h. The platform only required power for a heat source and showed potential for point of care diagnostics in resource-limited settings. For bacteria detection, anti-E. coli O157 functionalised magnetic beads were used to capture cells with > 90% efficiency and on-chip fluorescence in situ hybridisation and a staining assay were explored for bacteria identification.A wide variety of microfluidic approaches for pathogen analysis have been devised in the literature with different advantages and drawbacks. Careful evaluation based on their purpose, integrated steps and end user is critical. Input from stakeholders right from the start of a project and throughout is vital to success. The platforms investigated herein have potential for applications such as sample preparation, pathogen concentration and specific molecular detection of E. coli O157, N. gonorrhoeae DNA, and SARS-CoV-2 RNA. With further development and clinical validation, the widespread use of these systems could facilitate early diagnosis of infectious diseases, allowing timely management of outbreaks and treatment and slowing the incidence of antimicrobial resistance

Polymer Microsystems for the Enrichment of Circulating Tumor Cells and their Clinical Demonstration

Cancer research is centered on the discovery of new biomarkers that could unlock the obscurities behind the mechanisms that cause cancer or those associated with its spread (i.e., metastatic disease). Circulating tumor cells (CTCs) have emerged as attractive biomarkers for the management of many cancer-related diseases due primarily to the ease of securing them from a simple blood draw. However, their rarity (~1 CTC per mL of whole blood) makes enrichment analytically challenging. Microfluidic systems are viewed as exquisite platforms for the clinical analysis of CTCs due to their ability to be used in an automated fashion, minimizing sample loss and contamination. This has formed the basis of the reported research, which focused on the development of microfluidic systems for CTC analysis. The system reported herein consisted of a modular design and targeted the analysis of CTCs using pancreatic ductal adenocarcinoma (PDAC) as the model disease for determining the utility of the system. The system was composed of 3 functional modules; (i) a thermoplastic CTC selection module consisting of high aspect ratio (30 µm x 150 µm) channels; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for phenotype identification of selected CTCs. The system could exhaustively process 7.5 mL of blood in \u3c45 min with CTC recoveries \u3e90% directly from whole blood. In addition, significantly reduced assay turnaround times (8 h to 1.5 h) was demonstrated. We also show the ability to detect KRAS gene mutations from CTCs enriched by the microfluidic system. As a proof-of-concept, the ability to identify KRAS point mutations using a PCR/LDR/CE assay from as low as 10 CTCs enriched by the integrated microfluidic system was demonstrated. Finally, the clinical utility of the polymer-based microfluidic device for the analysis of circulating multiple myeloma cells (CMMCs) was demonstrated as well. Parameters such as translational velocity and recovery of CMMCs were optimized and found to be 1.1 mm/s and 71%, respectively. Also demonstrated was on-chip immunophenotyping and clonal testing of CMMCs, which has been reported to be prognostically significant. Further, a pilot study involving 26 patients was performed using the polymer microfluidic device with the aim of correlating the number of CMMCs with disease activity. An average of 347 CMMCs/mL of whole blood was recovered from blood volumes of approximately 0.5 mL

Imaging extracellular vesicles arising from apoptotic tumour cells for cancer diagnosis and monitoring

As a large part of all health-related research is focused on cancer, and with several diagnostic and therapeutic procedures continuously emerging, the fact that this disease remains mostly uncured often seems overwhelming. Cancer is a disease with extremely heterogenous causes and biologic backgrounds, and multiple mechanisms have been identified as cancer-promoting, acting on several stages of the tumour progression. Among numerous other networks, cancer cells use their own death in order to signal an urgency for survival to their neighbouring cells. It has been observed that while a cancer cell is undergoing apoptosis, it can release signals which upon receipt by surrounding cells can promote the growth of tumour. Apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data indicate that, besides its role in tumour suppression, it can also promote oncogenic proliferation. Highly aggressive tumours such as Burkitt Lymphoma (BL) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. The network of regeneration and tissue repair mechanisms driven by cell-death has been named as the “onco-regenerative niche” by our group, and it is hypothesized that amongst other elements, extracellular vesicles (EVs) are key mediators of apoptotic cell-derived tumour microenvironment signals. EVs are membrane delimited structures secreted by cells, containing multiple types of bioactive material, including markers of the tissue they originate from. They are released by almost all cells and during several phases of the cell life cycle. EVs show numerous applications in diagnostics, and there is an increasing interest in their biological functions. However, mainly because of their small size and heterogeneity, there are challenges associated with their analysis, and although EVs are gaining popularity in clinical diagnostic practice, the guidelines for analytic procedures have not been established to date. Because the vesicles are much smaller than cells and fall in the category of nanoparticles, the methods which can be applied for their analysis are dedicated to smaller entities or are special adaptations of other methods routinely used for larger particles such as cells. Here, we report on EVs released by apoptotic BL cells (Apo-EVs) in relation to their potential use as cancer biomarkers in lymphoma. The hypothesis of this project examines the Apo-EVs as to their distinct structural and biochemical characteristics which can be used in the context of disease diagnosis and monitoring. As Apo-EVs can reach the main blood circulation, the analysis of Apo-EVs in patients can provide with information about the stages and the progression of the disease. The two main axes this work move around on are firstly, the structural and biochemical analysis of the Apo-EVs in order to examine their special molecular characteristics which render those different from other EVs which are not related to apoptosis and secondly, the study of how Apo-EVs interact with cells present in the blood and whether their cargo can be transferred to the second. Those two sets of studies can provide a better understanding of Apo-EVs and their roles, aiming at contributing towards the development of a disease monitoring platform. This project is focused on analytical platforms and techniques which can be applied to the nano-scale for imaging EVs in pre-clinical research and with the potential for application on patient samples. In particular, EVs released in vitro by Burkitt Lymphoma cells undergoing apoptosis upon UV irradiation are used throughout this study. Basic physical properties of Apo-EVs such as structure, size distribution, surface charge and membrane fluidity are discussed using Cryo Electron Microscopy (EM) and tomography, Nanoparticle Tracking Analysis, Dynamic Light Scattering and fluorescence anisotropy respectively. For phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission EM, fluorescence microscopy and quantitative PCR. The second part of the analysis consists of a study of the interaction of Apo-EVs with blood components such as platelets, leukocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. For this purpose, cells and platelets from human blood were co-incubated with Apo-EVs in order to examine the uptake and the possibility of Apo-EV cargo delivery intracellularly. Looking at the differences between Apo- and non-Apo-EVs, the Apo-EVs have a larger diameter, while structurally, the two populations are not different. However, we have identified distinct Apo-EV markers such as active caspase 3 and histone-3, or DNA and small non-coding RNA-Y. There is also a strong interaction of EVs with platelets and leukocytes but not with red cells, indicating potential mechanisms of transfer of EV cargo in the circulation. It was also found that this interaction does not only concern the surface of the cells, but EVs can enter the platelets or cells, which supports the hypothesis that their special biochemical cargo can be transferred inside those cells. It is concluded that for the characterization of the heterogenous Apo-EV populations, comparison of results from of each method is essential for choosing the appropriate combination of analytical tools. Finally, we consider that the monitoring free circulating Apo-EV or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response

Microfluidic Selection of Aptamers towards Applications in Precision Medicine

Precision medicine represents a shift in medicine where large datasets are gathered for massive patient groups to draw correlations between disease cohorts. An individual patient can then be compared to these large datasets to determine the best treatment strategy. While electronic health records and next generation sequencing techniques have enabled much of the early applications for precision medicine, the human genome only represents a fraction of the information present and important to a person’s health. A person’s proteome (peptides and proteins) and glycome (glycans and glycosylation patterns) contain biomarkers that indicate health and disease; however, tools to detect and analyze such biomarkers remain scarce. Thus, precision medicine databases are lacking a major source of phenotypic data due to the absence of available methods to explore these domains, despite the potential of such data to allow further stratification of patients and individualized therapeutic strategies. Available methods to detect non-nucleic acid biomarkers are currently not well suited to address the needs of precision medicine. Mass spectrometry techniques, while capable of generating high throughput data, lack standardization, require extensive preparative steps, and have many sources of errors. Immunoassays rely on antibodies which are time consuming and expensive to produce for newly discovered biomarkers. Aptamers, analogous to antibodies but composed of nucleotides and isolated through in vitro methods, have potential to identify non-nucleic acid biomarkers but methods to isolate aptamers remain labor and resource intensive and time consuming. Recently, microfluidic technology has been applied to the aptamer discovery process to reduce the aptamer development time, while consuming smaller amounts of reagents. Methods have been demonstrated that employ capillary electrophoresis, magnetic mixers, and integrated functional chambers to select aptamers. However, these methods are not yet able to fully integrate the entire aptamer discovery process on a single chip and must rely on off-chip processes to identify aptamers. In this thesis, new approaches for aptamer selection are developed that aim to integrate the entire process for aptamer discovery on a single chip. These approaches are capable of performing efficient aptamer selection and polymerase chain reaction based amplification while utilizing highly efficient bead-based reactions. The approaches use pressure driven flow, electrokinetic flow or a combination of both to transfer aptamer candidates through multiple rounds of affinity selection and PCR amplification within a single microfluidic device. As such, the approaches are capable of isolating aptamer candidates within a day while consuming <500 µg of a target molecule. The utility of the aptamer discovery approach is then demonstrated with examples in precision medicine over a broad spectrum (small molecule to protein) of molecular targets. Seeking to demonstrate the potential of the device to generate probes capable of accessing the human glycome (an emerging source of precision medicine biomarkers), aptamers are isolated against gangliosides GM1, GM3, and GD3, and a glycosylated peptide. Finally, personalized, patient specific aptamers are isolated against a multiple myeloma patient serum sample. The aptamers have high affinity only for the patient derived antibody

Metal-organic framework (MOF) coatings for separation and biosensing

This dissertation is an interdisciplinary research project, which encompass the understanding of material science and surface engineering for MOF applications in separation and biosensing via three main studies. To expand the application of MOF in industry and commercial devices, the formation of continuous and intergrown MOF coating on flexible polymeric materials is necessary. It is found that the use of polydopamine/polyethyleneimine (PDA/PEI) coating for ZIF-8 nucleation and growth is highly dependent on the pre-treatment protocol, morphology, surface chemistry and surface charge of the modified support material. The main finding from this study is that the formation of dense PDA-PEI intermediate coating plays a key role in ensuring intergrown and connected polycrystalline ZIF-8 film on microporous polymeric support. It is found that not only the addition of biomaterials into the ZIF-8 structure were successful, the changes in the resulting morphology were also observed in terms of amorphous-crystalline structure, density, and roughness of ZIF materials. Next, biomineralization of GOx&HRP enzyme in MOF composite is utilized as a biosensor device. The results show that the ZIF-8/GOx&HRP in-situ composites have good acid and thermal stability compared to samples without ZIF-8. ZIF-8/GOx&HRP in-situ shows high selectivity towards glucose, linear sensitivity of 0.00303 Abs/μM. An unexpected benefit of this approach is the ability of the ZIF-8 thin film structure to provide a diffusion limiting effect for substrate influx; thus, producing high range of linear response range (8 µM-5mM of glucose). This study provides insight on the spatial location of the enzymes in MOF thin films, the effect of MOF functional group to enzyme activity and the potential of such patterning techniques for MOF-based biosensors using other types of biological elements such as antibodies and aptamers. Lastly, ZIF-8 membrane is fabricated using tannic acid and iron complexes (TA-Fe(III)) which is a cheaper, reversible, and green alternative to PDA/PEI and enzyme-induced MOF growth. The novel TA-Fe(III)/ZIF-8 film demonstrated ion selectivity ratio of K+/ Mg2+ (4.49), Na+/ Mg2+ (4.0) and Li+/ Mg2+ (3.87) which is excellent for lithium-ion extraction application from brine. Further investigation suggests that partial dehydration-hydration process plays a role for ion transport mechanism across the TA-Fe(III)/ZIF-8. The ion and gas separation performance of ZIF-8 based on TA-Fe(III) and PDA/PEI coating are compared to further discuss the role of surface modification on the quality of ZIF separation. The novelty of the dissertation lies in the new understanding of the interactions of support material, surface chemistry and MOF synthesis chemistry with guest molecules, which could open an avenue for large-scale manufacturing and commercialization of various MOF based devices